High concentration alpha-glucosidase compositions for the treatment of pompe disease

ABSTRACT

The present application provides for compositions comprising high concentrations of acid a-glucosidase in combination with an active site-specific chaperone for the acid α-glucosidase, and methods for treating Pompe disease in a subject in need thereof, that includes a method of administering to the subject such compositions. The present application also provides methods for increasing the in vitro and in vivo stability of an acid α-glucosidase enzyme formulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/015,556, filed Jun. 22, 2018, which is a divisional of U.S. patentapplication Ser. No. 15/192,137, filed Jun. 24, 2016, which is acontinuation of U.S. patent application Ser. No. 14/379,131, filed Aug.15, 2014, which is the U.S. national stage entry of PCT/US13/29660,filed Mar. 7, 2013, which claims the benefit of U.S. ProvisionalApplication No. 61/607,920, filed Mar. 7, 2012, and U.S. ProvisionalApplication No. 61/750,718, filed Jan. 9, 2013, to each of whichpriority is claimed and each of which are incorporated herein byreference in their entireties.

1. INTRODUCTION

The present invention relates to methods of treating, preventing, and/orameliorating Pompe Disease. The present invention also relates tocompositions and medicaments which may be labeled for use in thetreatment of Pompe Disease.

2. BACKGROUND OF THE INVENTION

Pompe disease (acid maltase deficiency) is caused by a deficiency in theenzyme acid α-glucosidase (GAA). GAA metabolizes glycogen, a storageform of sugar used for energy, into glucose. The accumulation ofglycogen leads to progressive muscle myopathy throughout the body whichaffects various body tissues, particularly the heart, skeletal muscles,liver, and nervous system. According to the National Institute ofNeurological Disorders and Stroke, Pompe disease is estimated to occurin about 1 in 40,000 births.

There are three recognized types of Pompe disease—infantile, juvenile,and adult onset (see, e.g., Hirschhom and Reuser, In: Scriver C R,Beaudet A L, Sly W, Valle D, editors; The Metabolic and Molecular Basesof Inherited Disease, Vol. III, New York: McGraw-Hill; 2001. p.3389-420, 2001: 3389-3420). Infantile-onset Pompe Disease is the mostsevere, and presents with symptoms that include severe lack of muscletone, weakness, enlarged liver and heart, and cardiomyopathy. Swallowingmay become difficult and the tongue may protrude and become enlarged.Most children die from respiratory or cardiac complications before theage of two, although a sub-set of infantile-onset patients live longer(non-classical infantile patients). Juvenile onset Pompe disease firstpresents in early to late childhood and includes progressive weakness ofthe respiratory muscles in the trunk, diaphragm, and lower limbs, aswell as exercise intolerance. Most juvenile onset Pompe patients do notlive beyond the second or third decade of life. Adult onset symptomsinvolve generalized muscle weakness and wasting of respiratory musclesin the trunk, lower limbs, and diaphragm. Some adult patients are devoidof major symptoms or motor limitations.

Unless identified during pre-natal screening, diagnosis of Pompe diseaseis a challenge. Diagnosis of adult-onset Pompe is even more difficultsince number, severity, and type of symptoms a patient experiences varywidely, and may suggest more common disorders such as musculardystrophies. Diagnosis is confirmed by measuring α-glucosidase activityand/or detecting pathologic levels of glycogen from biological samples.Currently the only approved therapy is enzyme replacement therapy withrecombinant α-glucosidase.

Pompe disease is one of several of glycogen pathologies. Others includeDebrancher deficiency (Cori's-Forbes' disease; Glycogenosis type III);Branching deficiency (Glycogenosis type IV; Andersen's disease);Myophsophorylase (McArdle's disease, Glycogen storage disease V);Phosphofructokinase deficiency-M isoform (Tauri's disease; Glycogenosistype VII); Phosphorylase b Kinase deficiency (Glycogenosis type VIII);Phosphoglycerate kinase A-isoform deficiency (Glycogenosis IX);Phosphoglycerate M-mutase deficiency (Glycogenosis type X).

3. SUMMARY OF THE INVENTION

The present invention relates to methods for the treatment of PompeDisease (e.g., infantile-onset Pompe disease), by administering to anindividual in need of such treatment an acid α-glucosidase (GAA) enzyme,(e.g., a recombinant human GAA (rhGAA)) in combination with an ActiveSite-Specific Chaperone (ASSC) for the GAA enzyme (e.g.,1-deoxynojirimycin (DNJ, 1-DNJ)).

The present invention further provides a method of increasing thestability of a GAA enzyme in a proper conformation, in vivo and invitro. In one embodiment, an acid α-glucosidase (GAA) enzyme (e.g., arecombinant human GAA (rhGAA)) in combination with an ASSC for the GAAenzyme (e.g., 1-deoxynojirimycin or 1-deoxynojirimycin-HCl) isadministered to an individual in need of such treatment. The GAA enzymeis stabilized conformationally when combined with an ASSC and iswell-suited to withstand, for example, thermal and pH challenges.

In certain embodiments, the GAA enzyme is combined with an ASSC at ahigh concentration, for example, at a concentration between about 5 andabout 250 mg/mL.

In certain embodiments, the GAA enzyme is combined with an ASSC at ahigh concentration, for example, at a concentration selected from thegroup consisting of about 25 mg/mL, about 80 mg/mL, about 115 mg/mL,about 160 mg/mL, about 200 mg/mL and about 240 mg/mL.

In certain embodiments, the GAA enzyme is combined with an ASSC, whereinthe ASSC is present at a concentration between about 5 mg/mL and about200 mg/mL.

In certain embodiments, the GAA enzyme is combined with an ASSC, whereinthe ASSC is present at a concentration selected from the groupconsisting of about 32 mg/mL and about 160 mg/mL.

In certain embodiments, the GAA enzyme is combined with an ASSC, whereinthe ASSC is present at a concentration between about 0.5 mM and about 20mM.

In certain embodiments, the GAA enzyme is combined with an ASSC as aco-formulation.

In certain embodiments, the GAA enzyme is combined with an ASSC in aco-formulation, wherein the co-formulation further comprises anexcipient. In certain embodiments, the excipient is selected from thegroup consisting of polyethylene glycol (PEG), PEG-400, arginine,arginine and glutamic acid, proline, gamma-cyclodextrin and combinationsthereof.

In certain embodiments, the formulations of the invention maintainphysical and chemical stability over extended periods despite the highconcentration of protein, and have a viscosity suitable for subcutaneousadministration. The formulations of the invention are established, atleast in part, on the surprising finding that a GAA enzyme combined withan ASSC can remain soluble at a high concentration (e.g., 25 mg/mL) andremain non-aggregated while maintaining a viscosity suitable forinjection (e.g., subcutaneous administration).

In certain embodiments, the compositions of the present inventioncomprise more than about 5 mg/mL of GAA enzyme.

In certain embodiments, the compositions of the invention comprise about25 mg/mL GAA enzyme and about 10 mM DNJ.

In certain embodiments, the compositions of the invention comprise 1 toabout 25 mg/mL GAA enzyme and about 1 mM DNJ.

An advantage of the formulation of the invention is that it provides ahigh concentration of protein without increased protein aggregation,which commonly occurs with increased protein concentration. In oneembodiment, the formulation of the invention has less than about 1%aggregate protein.

According to one aspect of the invention, methods of enhancing deliveryof GAA to tissues, for example muscle tissue, of an individual withPompe disease are provided. The methods include administering GAA incombination with an ASSC subcutaneously to the individual. In someembodiments, the GAA in combination with an ASSC is administered in asufficient dose to result in a peak concentration of GAA in tissues ofthe subject within about 24 hours after the administration of the dose.In certain embodiments, the GAA in combination with an ASSC isadministered in a sufficient dose to result in a peak concentration ofGAA in tissues of the subject within about 10 to about 50 hours, orabout 45, 40, 35, 30, 25, or fewer hours after the administration of thedose. In some embodiments, the dose does not result in a toxic level ofGAA in the liver of the individual.

In various non-limiting embodiments, the ASSC for the GAA enzyme is asmall molecule inhibitor of the GAA enzyme, including reversiblecompetitive inhibitors of the GAA enzyme.

In one embodiment the ASSC is represented by the formula:

where R₁ is H or a straight or branched alkyl, cycloalkyl, alkoxyalkylor aminoalkyl containing 1-12 carbon atoms optionally substituted withan —OH, —COOH, —Cl, —F, —CF₃, —OCF₃, —O—C(═O)N-(alkyl)₂; and R₂ is H ora straight or branched alkyl, cycloalkyl, or alkoxylalkyl containing 1-9carbon atoms; including pharmaceutically acceptable salts, esters andprodrugs thereof. In one embodiment, the ASSC is as defined above, withR₁ being H. In another embodiment, the ASSC is as defined above, with R₂being H.

In one particular non-limiting embodiment, the ASSC is1-deoxynojirimycin (1-DNJ), which is represented by the followingformula:

or a pharmaceutically acceptable salts, esters or prodrug of1-deoxynojirimycin. In one embodiment, the salt is hydrochloride salt(i.e. 1-deoxynojirimycin-HCl).

In one particular non-limiting embodiment, the ASSC isN-butyl-deoxynojirimycin (NB-DNJ; Zavesca®, Actelion PharmaceuticalsLtd, Switzerland), which is represented by the following formula:

or a pharmaceutically acceptable salt, ester or prodrug of NB-DNJ.

In one particular non-limiting embodiment, the ASSC is C₁₀H₁₉NO₄, whichis represented by the following formula:

or a pharmaceutically acceptable salt, ester or prodrug of C₁₀H₁₉NO₄. Inone embodiment, the salt is hydrochloride salt.

In one particular non-limiting embodiment, the ASSC is C₁₂H₂₃NO₄, whichis represented by the following formula:

or a pharmaceutically acceptable salt, ester or prodrug of C₁₂H₂₃NO₄. Inone embodiment, the salt is hydrochloride salt.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the stability of recombinant human GAA (Myozyme, GenzymeCorp.) at ER pH (7.4) or lysosomal pH (5.2) in the presence or absenceof 100 μM of 1-deoxynojirimycin hydrochloride (1-DNJ-HCl) as determinedin a thermal stability assay. The thermal stability assay utilizes heatto induce protein denaturation, which is monitored using a SYPRO Orangedye that fluoresces upon binding to hydrophobic amino acids (which arenot exposed in a folded protein). A protein structure that requires moreheat to denature is by definition more stable. As shown above, Myozyme®is ordinarily much more stable at lysosomal pH (5.2) than at ER pH(7.4). However, the enzyme stability at pH 7.4 is significantlyincreased upon addition of 100 μM of 1-deoxynojirimycin, as compared toMyozyme® alone.

FIG. 2A depicts the effects of 1-DNJ-HCl on recombinant human GAA(Myozyme®, Genzyme Corp.) enzymatic activity at plasma pH (7.4) orlysosomal pH (5.2) at 37° C. GAA activity was evaluated to assess theability of an ASSC of GAA to prolong the activity of the rhGAA overtime. Myozyme® (45 nM) was incubated in pH 7.4 or pH 5.2 buffer with orwithout 50 μM 1-DNJ at 37° C. over 24 hours. Samples were assayed forGAA enzyme activity using 4-MU-α-glucose at 0, 3, 6 and 24 hours and theresidual GAA activity was expressed as % of initial activity. Theseresults indicate that 1-DNJ ameliorates the loss of GAA enzyme activityat plasma pH (7.4).

FIG. 2B depicts a parallel SYPRO Orange thermal stability experiment todetermine if the loss of enzyme activity shown in FIG. 2A, particularlythe loss of Myozyme® activity at ER pH (7.4), correlates with proteinunfolding and denaturation. Myozyme® (0.9 μM) was incubated in pH 7.4 orpH 5.2 buffer with or without 100 μM 1-DNJ-HCl at 37° C. and the proteinfolding was monitored every hour over 24 hours. FIGS. 2A and 2B showthat GAA denaturation correlates with loss of enzyme activity (comparecurve with diamond curves in the two figures). More importantly, theseresults indicate that 1-DNJ can prevent GAA denaturation and loss ofenzyme activity at plasma pH.

FIG. 3 depicts the results of GAA activity tests on GAA KO MiceReceiving ERT with and without concurrent oral administration of1-DNJ-HCl. Myozyme® was administered via IV infusion at a dose of 10mg/kg, once per week for up to 3 weeks either alone or in combinationwith 10, 100, or 1000 mg/kg of 1-DNJ-HCl 30 min prior to, and 8, 16, and24 hours post-Myozyme® administration. These results demonstrate thatMyozyme® tissue uptake (as a measure of GAA activity) declined at 7 dayspost injection. Coadministration of 1-DNJ-HCl with Myozyme® facilitateda dose-dependent increase in Myozyme® uptake for up to 7 days postinjection. The effect of 1-DNJ-HCl was more pronounced and significant(p<0.05 t-test vs. Myozyme® alone) at 4 and 7 days post injection ofeither 1, 2, or 3 weekly infusions of Myozyme®.

FIG. 4 demonstrates that 1-DNJ-HCl inhibits GAA with an IC₅₀ of about 1μM.

FIG. 5 depicts the results of a thermal stability assay that utilizesheat to induce protein denaturation, which is monitored using a SYPROOrange dye that fluoresces upon binding to hydrophobic amino acids(which are not exposed in a folded protein). 1-DNJ-HCl increases GAAthermostability as evident by increases in GAA's melting temperature ina dose-dependent manner.

FIG. 6 depicts the results of GAA activity in rats over 24 hours afterIV administration of 10 mg/kg of rhGAA or saline with and without 3mg/kg or 30 mg/kg of orally administered 1-DNJ-HCl. The rhGAA or salinewas administered 30 minutes after administration of the 1-DNJ-HCl. Inthis example, the 1-DNJ-HCl inhibited the loss of enzyme activitypost-administration, thereby increasing the in vivo half life of rhGAA.The in vivo half life of rhGAA increased from 1.4±0.2 hours (0 mg/kg of1-DNJ-HCl) to 2.1±0.2 hours (3 mg/kg of 1-DNJ-HCl) and 3.0±0.4 hours (30mg/kg of 1-DNJ-HCl).

FIGS. 7A-7B depict the GAA activity in heart (A) and diaphragm tissue(B) for ERT monotherapy and ERT/ASSC co-therapy (rhGAA+1-DNJ-HCl) whenadministered to a GAA KO mouse. rhGAA uptake in the heart and diaphragmis increased when co-administered with 1-DNJ-HCl.

FIG. 8 shows that 1-DNJ-HCl prevents rhGAA enzyme inactivation in blood.Myozyme (0.5 μM) was incubated at 37° C. in citrate anti-coagulatedwhole blood in the presence or absence of 50 μM 1-DNJ-HCl. Aliquots werecollected at 0, 2, 4, 8 and 24 hrs and centrifuged to obtain plasma.These plasma samples were then diluted in potassium acetate buffer (pH4.0) and assayed for GAA activity using the4-methylumbeliferyl-α-glucose (4-MUG) fluorogenic substrate. Themeasured GAA activity for individual samples at each time point wasnormalized to the 0 hr and expressed as % of initial activity. Data from4 independent experiments were analyzed to obtain the mean (and standarddeviation) and plotted versus time to assess the loss of enzyme activityover this time course.

FIG. 9 shows that low 1-DNJ-HCl concentrations prevent rhGAA enzymeinactivation in blood. Myozyme® (0.5 μM) was incubated at 37° C. incitrate anti-coagulated whole blood with varying 1-DNJ-HClconcentrations (0-100 μM). Aliquots were collected at 0, 3 and 6 hrs andcentrifuged to obtain plasma. These plasma samples were then diluted inpotassium acetate buffer (pH 4.0) and assayed for GAA activity using the4-methylumbeliferyl-α-glucose (4-MUG) fluorogenic substrate. Themeasured GAA activity for individual samples at each time point wasnormalized to the 0 hr and expressed as % of initial activity. Theresidual GAA enzyme activity was plotted versus time to assess the lossof enzyme activity with respect to 1-DNJ-HCl concentration over thistime course.

FIG. 10 shows the experimental design for Example 9.

FIGS. 11A-11D show that Myozyme® co-administered with 1-DNJ-HCl resultedin significantly greater tissue glycogen reduction in GAA KO mice ascompared to Myozyme® alone. Glycogen reduction with Myozyme® alone was93±1%, 41±4%, 69±3%, and 18±4%, in heart (A), diaphragm (B), soleus (C),and quadriceps (D), respectively, relative to untreated mice. Glycogenreduction with Myozyme® co-administered with 1-DNJ-HCl was 96±0.6%,66±5%, 82±3%, and 23±3%, respectively.

FIG. 12 shows that combining 1 mM DNJ with 25 mg/mL Myozyme® reducesaggregation of Myozyme® at neutral pH 7.4 and 37° C. Aggregation wasassessed after a 4 week incubation period.

FIG. 13 shows that administering 30 mg/kg DNJ co-formulated with anamount equivalent to 10 mg/kg Myozyme® via tail vein injection increasedthe circulating plasma half-life and tissue uptake of Myozyme® inquadriceps.

FIG. 14 shows that subcutaneous administration of DNJ co-formulated with20 mg/kg Myozyme® increased the circulating levels of rhGAA compared toadministration of 20 mg/kg Myozyme® without DNJ.

FIG. 15 shows rhGAA activity in skin at the injection site 3 daysfollowing the last subcutaneous administration of rhGAA or aco-formulation of rhGAA and 1-DNJ to mouse, as described in Example 13.

FIGS. 16A-16B show rhGAA activity (A) and glycogen level (B) in ventralskin 3 days (A) and 14 days (B) following the last subcutaneousadministration of rhGAA or a co-formulation of rhGAA and 1-DNJ to mouse,as described in Example 13.

FIGS. 17A-17B show rhGAA activity (A) and glycogen level (B) in heart 3days (A) and 14 days (B) following the last subcutaneous administrationof rhGAA or a co-formulation of rhGAA and 1-DNJ to mouse, as describedin Example 13.

FIGS. 18A-18B show rhGAA activity (A) and glycogen level (B) in tongue 3days (A) and 14 days (B) following the last subcutaneous administrationof rhGAA or a co-formulation of rhGAA and 1-DNJ to mouse, as describedin Example 13.

FIGS. 19A-19B show rhGAA activity (A) and glycogen level (B) indiaphragm 3 days (A) and 14 days (B) following the last subcutaneousadministration of rhGAA or a co-formulation of rhGAA and 1-DNJ to mouse,as described in Example 13.

FIGS. 20A-20B show rhGAA activity (A) and glycogen level (B) in biceps 3days (A) and 14 days (B) following the last subcutaneous administrationof rhGAA or a co-formulation of rhGAA and 1-DNJ to mouse, as describedin Example 13.

FIGS. 21A-21B show rhGAA activity (A) and glycogen level (B) in triceps3 days (A) and 14 days (B) following the last subcutaneousadministration of rhGAA or a co-formulation of rhGAA and 1-DNJ to mouse,as described in Example 13.

FIGS. 22A-22B show rhGAA activity (A) and glycogen level (B) ingastrocnemius 3 days (A) and 14 days (B) following the last subcutaneousadministration of rhGAA or a co-formulation of rhGAA and 1-DNJ to mouse,as described in Example 13.

FIGS. 23A-23B show rhGAA activity (A) and glycogen level (B) inquadriceps 3 days (A) and 14 days (B) following the last subcutaneousadministration of rhGAA or a co-formulation of rhGAA and 1-DNJ to mouse,as described in Example 13.

FIG. 24 shows rhGAA activity in soleus 3 days following the lastsubcutaneous administration of rhGAA or a co-formulation of rhGAA and1-DNJ to mouse, as described in Example 13.

FIG. 25 shows rhGAA activity in liver 3 days following the lastsubcutaneous administration of rhGAA or a co-formulation of rhGAA and1-DNJ to mouse, as described in Example 13.

FIG. 26 shows a comparison of rhGAA activity in the various tissuestested 3 days following the last subcutaneous administration of rhGAA ora co-formulation of rhGAA and 1-DNJ to mouse, as described in Example13.

FIGS. 27A-27B show plasma rhGAA activity 2 hours (A) and 4 hours (B)following the last subcutaneous administration of rhGAA or aco-formulation of rhGAA and 1-DNJ to mouse, as described in Example 13.

FIGS. 28A-28B show plasma rhGAA activity (B) and plasma GAA proteinlevel (as measured by Western blot) (A) 2 hours following the lastsubcutaneous administration of rhGAA or a co-formulation of rhGAA and1-DNJ to mouse, as described in Example 13.

FIGS. 29A-29B show plasma rhGAA activity (B) and plasma GAA proteinlevel (as measured by Western blot) (A) 4 hours following the lastsubcutaneous administration of rhGAA or a co-formulation of rhGAA and1-DNJ to mouse, as described in Example 13.

5. DETAILED DESCRIPTION

The present invention is based at least in part on the discovery thatcombining an acid α-glucosidase (GAA) enzyme (e.g., a recombinant humanGAA (rhGAA)), with an ASSC for the GAA enzyme (e.g.,1-deoxynojirimycin), results in a surprising increase in GAA activity invivo as compared to either treatment alone. The present invention isalso based at least in part on the discovery that a GAA enzyme (e.g.,rhGAA) stabilizes a proper conformation—both in vitro and in vivo—uponaddition of an ASSC for the GAA enzyme. The present invention is alsobased at least in part on the discovery that combining an ASSC with ahigh concentration of GAA reduces GAA aggregation, which commonly occurswith increased GAA concentration.

For clarity and not by way of limitation, this detailed description isdivided into the following sub-portions:

-   -   (i) Definitions;    -   (ii) Pompe Disease;    -   (iii) Obtaining GAA and ASSC;    -   (iv) Treatment of Pompe Disease with ERT and an ASSC;    -   (v) Pharmaceutical Compositions;    -   (vi) In Vitro Stability; and    -   (vii) In Vivo Stability.

5.1 Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

According to the invention, a “subject” or “patient” is a human ornon-human animal. Although the animal subject is preferably a human, thecompounds and compositions of the invention have application inveterinary medicine as well, e.g., for the treatment of domesticatedspecies such as canine, feline, and various other pets; farm animalspecies such as bovine, equine, ovine, caprine, porcine, etc.; wildanimals, e.g., in the wild or in a zoological garden; and avian species,such as chickens, turkeys, quail, songbirds, etc.

The term “enzyme replacement therapy” or “ERT” refers to refers to theintroduction of a non-native, purified enzyme into an individual havinga deficiency in such enzyme. The administered enzyme can be obtainedfrom natural sources or by recombinant expression. The term also refersto the introduction of a purified enzyme in an individual otherwiserequiring or benefiting from administration of a purified enzyme, e.g.,suffering from protein insufficiency. The introduced enzyme may be apurified, recombinant enzyme produced in vitro, or enzyme purified fromisolated tissue or fluid, such as, e.g., placenta or animal milk, orfrom plants.

The term “stabilize a proper conformation” refers to the ability of acompound or peptide or other molecule to associate with a wild-typeprotein, or to a mutant protein that can perform its wild-type functionin vitro and in vivo, in such a way that the structure of the wild-typeor mutant protein can be maintained as its native or proper form. Thiseffect may manifest itself practically through one or more of (i)increased shelf-life of the protein; (ii) higher activity perunit/amount of protein; or (iii) greater in vivo efficacy. It may beobserved experimentally through increased yield from the ER duringexpression; greater resistance to unfolding due to temperature increases(e.g., as determined in thermal stability assays), or the present ofchaotropic agents, and by similar means.

As used herein, the term “active site” refers to the region of a proteinthat has some specific biological activity. For example, it can be asite that binds a substrate or other binding partner and contributes theamino acid residues that directly participate in the making and breakingof chemical bonds. Active sites in this invention can encompasscatalytic sites of enzymes, antigen biding sites of antibodies, ligandbinding domains of receptors, binding domains of regulators, or receptorbinding domains of secreted proteins. The active sites can alsoencompass transactivation, protein-protein interaction, or DNA bindingdomains of transcription factors and regulators.

As used herein, the term “active site-specific chaperone” refers to anymolecule including a protein, peptide, nucleic acid, carbohydrate, etc.that specifically interacts reversibly with an active site of a proteinand enhances formation of a stable molecular conformation. As usedherein, “active site-specific chaperone” does not include endogenousgeneral chaperones present in the ER of cells such as Bip, calnexin orcalreticulin, or general, non-specific chemical chaperones such asdeuterated water, DMSO, or TMAO.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 95% pure; morepreferably, at least 97% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art. In a specific embodiment, purified means that thelevel of contaminants is below a level acceptable to regulatoryauthorities for safe administration to a human or non-human animal.

As used herein, the terms “mutant” and “mutation” mean any detectablechange in genetic material, e.g., DNA, or any process, mechanism orresult of such a change. This includes gene mutations, in which thestructure (e.g., DNA sequence) of a gene is altered, any gene or DNAarising from any mutation process, and any expression product (e.g.,RNA, protein or enzyme) expressed by a modified gene or DNA sequence.

As used herein the term “mutant protein” refers to proteins translatedfrom genes containing genetic mutations that result in altered proteinsequences. In a specific embodiment, such mutations result in theinability of the protein to achieve its native conformation under theconditions normally present in the ER. The failure to achieve thisconformation results in these proteins being degraded, rather than beingtransported through their normal pathway in the protein transport systemto their proper location within the cell. Other mutations can result indecreased activity or more rapid turnover.

As used herein the term “wild-type gene” refers to a nucleic acidsequences which encodes a protein capable of having normal biologicalfunctional activity in vivo. The wild-type nucleic acid sequence maycontain nucleotide changes that differ from the known, publishedsequence, as long as the changes result in amino acid substitutionshaving little or no effect on the biological activity. The termwild-type may also include nucleic acid sequences engineered to encode aprotein capable of increased or enhanced activity relative to theendogenous or native protein.

As used herein, the term “wild-type protein” refers to any proteinencoded by a wild-type gene that is capable of having functionalbiological activity when expressed or introduced in vivo. The term“normal wild-type activity” refers to the normal physiological functionof a protein in a cell. Such functionality can be tested by any meansknown to establish functionality of a protein.

The term “genetically modified” refers to cells that express aparticular gene product following introduction of a nucleic acidcomprising a coding sequence which encodes the gene product, along withregulatory elements that control expression of the coding sequence.Introduction of the nucleic acid may be accomplished by any method knownin the art including gene targeting and homologous recombination. Asused herein, the term also includes cells that have been engineered toexpress or overexpress an endogenous gene or gene product not normallyexpressed by such cell, e.g., by gene activation technology.

The phrase “pharmaceutically acceptable”, whether used in connectionwith the pharmaceutical compositions of the invention, refers tomolecular entities and compositions that are physiologically tolerableand do not typically produce untoward reactions when administered to ahuman. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the compound is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils. Water oraqueous solution saline solutions and aqueous dextrose and glycerolsolutions are preferably employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition.

The terms “therapeutically effective dose” and “effective amount” referto the amount of the compound that is sufficient to result in atherapeutic response. In embodiments where an ASSC and GAA areadministered in a complex, the terms “therapeutically effective dose”and “effective amount” may refer to the amount of the complex that issufficient to result in a therapeutic response. A therapeutic responsemay be any response that a user (e.g., a clinician) will recognize as aneffective response to the therapy. Thus, a therapeutic response willgenerally be an amelioration of one or more symptoms or sign of adisease or disorder.

It should be noted that a concentration of the ASSC that is inhibitoryduring in vitro production, transportation, or storage of the purifiedtherapeutic protein may still constitute an “effective amount” forpurposes of this invention because of dilution (and consequent shift inbinding due to the change in equilibrium), bioavailability andmetabolism of the ASSC upon administration in vivo.

The term ‘alkyl’ refers to a straight or branched hydrocarbon groupconsisting solely of carbon and hydrogen atoms, containing nounsaturation, and which is attached to the rest of the molecule by asingle bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl).

The term “alkenyl” refers to a C₂-C₂₀ aliphatic hydrocarbon groupcontaining at least one carbon-carbon double bond and which may be astraight or branched chain, e.g., ethenyl, 1-propenyl, 2-propenyl(allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl.

The term “cycloalkyl” denotes an unsaturated, non-aromatic mono- ormulticyclic hydrocarbon ring system such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl. Examples of multicyclic cycloalkyl groupsinclude perhydronapththyl, adamantyl and norbornyl groups bridged cyclicgroup or sprirobicyclic groups, e.g., spiro (4,4) non-2-yl.

The term “aryl” refers to aromatic radicals having in the range of about6 to about 14 carbon atoms such as phenyl, naphthyl, tetrahydronapthyl,indanyl, biphenyl.

The term “heterocyclic” refers to a stable 3- to 15-membered ringradical which consists of carbon atoms and from one to five heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur. Forpurposes of this invention, the heterocyclic ring radical may be amonocyclic or bicyclic ring system, which may include fused or bridgedring systems, and the nitrogen, carbon, oxygen or sulfur atoms in theheterocyclic ring radical may be optionally oxidized to variousoxidation states. In addition, a nitrogen atom, where present, may beoptionally quaternized; and the ring radical may be partially or fullysaturated (i.e., heteroaromatic or heteroaryl aromatic).

The heterocyclic ring radical may be attached to the main structure atany heteroatom or carbon atom that results in the creation of a stablestructure.

The term “heteroaryl” refers to a heterocyclic ring wherein the ring isaromatic.

The substituents in the ‘substituted alkyl’, ‘substituted alkenyl’,‘substituted cycloalkyl’, ‘substituted aryl’ and ‘substitutedheteroaryl’ may be the same or different, with one or more selected fromthe groups hydrogen, halogen, acetyl, nitro, carboxyl, oxo (═O), CF₃,—OCF₃, NH₂, —C(═O)-alkyl₂, OCH₃, or optionally substituted groupsselected from alkyl, alkoxy and aryl.

The term “halogen” refers to radicals of fluorine, chlorine, bromine andiodine.

5.2 Pome Disease

Pompe disease is an autosomal recessive LSD characterized by deficientacid alpha glucosidase (GAA) activity which impairs lysosomal glycogenmetabolism. The enzyme deficiency leads to lysosomal glycogenaccumulation and results in progressive skeletal muscle weakness,reduced cardiac function, respiratory insufficiency, and/or CNSimpairment at late stages of disease. Genetic mutations in the GAA generesult in either lower expression or produce mutant forms of the enzymewith altered stability, and/or biological activity ultimately leading todisease. (see generally Hirschhom R, 1995, Glycogen Storage Disease TypeII: Acid α-Glucosidase (Acid Maltase) Deficiency, The Metabolic andMolecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill,New York, 7th ed., pages 2443-2464). The three recognized clinical formsof Pompe disease (infantile, juvenile and adult) are correlated with thelevel of residual α-glucosidase activity (Reuser A J et al., 1995,Glycogenosis Type II (Acid Maltase Deficiency), Muscle & NerveSupplement 3, S61-S69). ASSC (also referred to elsewhere as“pharmacological chaperones”) represent a promising new therapeuticapproach for the treatment of genetic diseases, such as lysosomalstorage disorders (e.g., Pompe Disease).

Infantile Pompe disease (type I or A) is most common and most severe,characterized by failure to thrive, generalized hypotonia, cardiachypertrophy, and cardiorespiratory failure within the second year oflife. Juvenile Pompe disease (type II or B) is intermediate in severityand is characterized by a predominance of muscular symptoms withoutcardiomegaly. Juvenile Pompe individuals usually die before reaching 20years of age due to respiratory failure. Adult Pompe disease (type IIIor C) often presents as a slowly progressive myopathy in the teenageyears or as late as the sixth decade (Felice K J et al., 1995, ClinicalVariability in Adult-Onset Acid Maltase Deficiency: Report of AffectedSibs and Review of the Literature, Medicine 74, 131-135).

In Pompe, it has been shown that α-glucosidase is extensively modifiedpost-translationally by glycosylation, phosphorylation, and proteolyticprocessing. Conversion of the 110 kilodalton (kDa) precursor to 76 and70 kDa mature forms by proteolysis in the lysosome is required foroptimum glycogen catalysis.

As used herein, the term “Pompe Disease” refers to all types of PompeDisease. The formulations and dosing regimens disclosed in thisapplication may be used to treat, for example, Type I, Type II or TypeIII Pompe Disease.

5.3 Obtaining GAA and ASSC

GAA may be obtained from a cell endogenously expressing the GAA, or theGAA may be a recombinant human GAA (rhGAA), as described herein. In one,non-limiting embodiment, the rhGAA is a full length wild-type GAA. Inother non-limiting embodiments, the rhGAA comprises a subset of theamino acid residues present in a wild-type GAA, wherein the subsetincludes the amino acid residues of the wild-type GAA that form theactive site for substrate binding and/or substrate reduction. As such,the present invention contemplates an rhGAA that is a fusion proteincomprising the wild-type GAA active site for substrate binding and/orsubstrate reduction, as well as other amino acid residues that may ormay not be present in the wild type GAA.

GAA may be obtained from commercial sources or may be obtained bysynthesis techniques known to a person of ordinary skill in the art. Thewild-type enzyme can be purified from a recombinant cellular expressionsystem (e.g., mammalian cells or insect cells-see generally U.S. Pat.No. 5,580,757 to Desnick et al.; U.S. Pat. Nos. 6,395,884 and 6,458,574to Selden et al.; U.S. Pat. No. 6,461,609 to Calhoun et al.; U.S. Pat.No. 6,210,666 to Miyamura et al.; U.S. Pat. No. 6,083,725 to Selden etal.; U.S. Pat. No. 6,451,600 to Rasmussen et al.; U.S. Pat. No.5,236,838 to Rasmussen et al.; and U.S. Pat. No. 5,879,680 to Ginns etal.), human placenta, or animal milk (see U.S. Pat. No. 6,188,045 toReuser et al.). After the infusion, the exogenous enzyme is expected tobe taken up by tissues through non-specific or receptor-specificmechanism. In general, the uptake efficiency (without use of an ASSC) isnot high, and the circulation time of the exogenous protein is short(Ioannu et al., Am. J. Hum. Genet. 2001; 68: 14-25). In addition, theexogenous protein is unstable and subject to rapid intracellulardegradation in vitro.

Other synthesis techniques for obtaining GAA suitable for pharmaceuticalmay be found, for example, in U.S. Pat. Nos. 7,560,424 and 7,396,811 toLebowitz et al., U.S. Published Application Nos. 2009/0203575,2009/0029467, 2008/0299640, 2008/0241118, 2006/0121018, and 2005/0244400to Lebowitz et al., U.S. Pat. Nos. 7,423,135, 6,534,300, and 6,537,785;International Published Application No. 2005/077093 and U.S. PublishedApplication Nos. 2007/0280925, and 2004/0029779. These references arehereby incorporated by reference in their entirety.

In one embodiment, the GAA is alglucosidase alfa, which consists of thehuman enzyme acid α-glucosidase (GAA), encoded by the most predominantof nine observed haplotypes of this gene and is produced by recombinantDNA technology in a Chinese hamster ovary cell line. Alglucosidase alphais available as Myozyme® and Lumizyme®, from Genzyme Corporation(Cambridge, Mass.).

ASSC may be obtained using synthesis techniques known to one of ordinaryskill in the art. For example, ASSC that may be used in the presentapplication, such as 1-DNJ may be prepared as described in U.S. Pat.Nos. 6,274,597 and 6,583,158, and U.S. Published Application No.2006/0264467, each of which is hereby incorporated by reference in itsentirety.

In one embodiment of the present application, the ASSC isα-homonojirimycin and the GAA is hrGAA (e.g., Myozyme® or Lumizyme®). Inan alternative embodiment the ASSC is castanospermine and the GAA ishrGAA (e.g., Myozyme® or Lumizyme®). The ASSC (e.g. α-homonojirimycinand castanospermine) may be obtained from synthetic libraries (see,e.g., Needels et al., Proc. Natl. Acad. Sci. USA 1993; 90:10700-4;Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 1993; 90:10922-10926; Lam etal., PCT Publication No. WO 92/00252; Kocis et al., PCT Publication No.WO 94/28028) which provide a source of potential ASSC's according to thepresent invention. Synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), andMicrosource (New Milford, Conn.). A rare chemical library is availablefrom Aldrich (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch(NC), or are readily producible. Additionally, natural and syntheticallyproduced libraries and compounds are readily modified through Res. 1986;155:119-29.

In one embodiment, ASSC's useful for the present invention areinhibitors of lysosomal enzymes and include glucose and galactoseimino-sugar derivatives as described in Asano et al., J. Med. Chem.1994; 37:3701-06; Dale et al., Biochemistry 1985; 24:3530-39; Goldman etal., J. Nat. Prod. 1996; 59:1137-42; Legler et al, Carbohydrate Res.1986; 155:119-29. Such derivatives include those that can be purchasedfrom commercial sources such as Toronto Research Chemicals, Inc. (NorthYork, On. Canada) and Sigma.

5.4 Treatment of Pompe Disease with ERT and an ASSC

In accordance with the invention, there are provided methods of usingGAA (e.g. rhGAA) in combination with an ASSC for the GAA. One embodimentof the present invention provides for combination therapy of GAA (e.g.hrGAA ERT) and an ASSC. For example, the ASSC chaperone1-deoxynojirimycin-HCl binds to mutant GAA and increases the ability ofthe GAA to stabilize to a proper conformation.

One embodiment of the present invention provides a method for treatingPompe subset patients with the IVS 1 (−13 T>G) splicing defect with anASSC and hrGAA enzyme replacement therapy. In cell lines derived fromlate-onset Pompe patients with this common splicing mutation,1-deoxynojirimycin-HCl increased GAA levels alone and in combinationwith hrGAA.

In one non-limiting embodiment of the present invention,1-deoxynojirimycin-HCl, or a pharmaceutically acceptable salt thereof,can be administered to a subject in a dose of between about 10 mg/kg to1000 mg/kg, preferably administered orally, either prior to, concurrentwith, or after administration of the GAA. In one non-limitingembodiment, 1-deoxynojirimycin-HCl and recombinant human GAA showsurprising efficacy on cellular enzyme activity, glycogen reduction andthe treatment of Pompe disease. In rats, the plasma half-life ofrecombinant human GAA (rhGAA) increased 2-fold when1-deoxynojirimycin-HCl (30 mg/kg p.o.) was administered in a dosingregimen that includes dosing 30 minutes prior to rhGAA injection. In GAAKO mice, the uptake of rhGAA was increased approximately 2-fold in heartand diaphragm when 1-deoxynojirimycin-HCl (100 mg/kg p.o.) was in adosing regimen that includes administration prior to rhGAA injection.These results indicate that co-administration of a an ASSC with rhGAAincrease the enzyme's exposure and tissue uptake in vivo in surprisingamounts.

For example, one embodiment of the present invention provides a methodof treating Pompe Disease comprising administering GAA (e.g. rhGAA)bi-weekly, weekly or once per two weeks for up to about 10 weeks incombination with from about 1 to about 5000 mg/kg of an ASSC (e.g.,1-DNJ-HCl) prior to, and in regular intervals after, the GAA infusion.For example, the ASSC could be administered within two hours of theinfusion, and then administered at regular intervals once, twice,three-times, four-times, five-times or six-times within 24 hourspost-infusion.

In one particular embodiment, the GAA is Myozyme® and is administeredvia infusion once per week and the ASSC (e.g., 1-DNJ-HCl) isadministered at 10 mg/kg, 100 mg/kg or 1000 mg/kg 30 minutes prior toinfusion, and then 8, 16, and 24 hours after each Myozyme® infusion.

In other particular embodiments, the GAA is Lumizyme® and isadministered via infusion once per week and the ASSC (e.g., 1-DNJ-HCl)is administered at 10 mg/kg, 100 mg/kg or 1000 mg/kg 30 minutes prior toinfusion, and then 8, 16, and 24 hours after each Lumizyme® infusion.

While not being bound by any particular theory, it is believed that acidα-glucosidase (GAA) functions to remove terminal glucose residues fromlysosomal glycogen. Some genetic mutations reduce GAA trafficking andmaturation. The pharmacological chaperone 1-DNJ increases GAA levels byselectively binding and stabilizing the enzyme in a proper conformationwhich restores proper protein trafficking to the lysosome.

In alternative embodiments, the ASSC is administered as described inInternational Publication No. 2008/134628, which is hereby incorporatedby reference in its entirety.

In some embodiments, the route of administration is subcutaneous.

Other routes of administration may be oral or parenteral, includingintravenous, intra-arterial, intraperitoneal, ophthalmic, intramuscular,buccal, rectal, vaginal, intraorbital, intracerebral, intradermal,intracranial, intraspinal, intraventricular, intrathecal,intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal,transdermal, or via inhalation. Intrapulmonary delivery methods,apparatus and drug preparation are described, for example, in U.S. Pat.Nos. 5,785,049, 5,780,019, and 5,775,320, each incorporated herein byreference. In some embodiments, the method of intradermal delivery is byiontophoretic delivery via patches; one example of such delivery istaught in U.S. Pat. No. 5,843,015, which is incorporated herein byreference.

Administration may be by periodic injections of a bolus of thepreparation, or as a sustained release dosage form over long periods oftime, or by intravenous or intraperitoneal administration, for example,from a reservoir which is external (e.g., an IV bag) or internal (e.g.,a bioerodable implant, a bioartificial organ, or a population ofimplanted GAA production cells). See, e.g., U.S. Pat. Nos. 4,407,957 and5,798,113, each incorporated herein by reference. Intrapulmonarydelivery methods and apparatus are described, for example, in U.S. Pat.Nos. 5,654,007, 5,780,014, and 5,814,607, each incorporated herein byreference. Other useful parenteral delivery systems includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, pump delivery, encapsulated cell delivery, liposomaldelivery, needle-delivered injection, needle-less injection, nebulizer,aeorosolizer, electroporation, and transdermal patch. Needle-lessinjector devices are described in U.S. Pat. Nos. 5,879,327; 5,520,639;5,846,233 and 5,704,911, the specifications of which are hereinincorporated by reference. Any of the GAA preparation described hereincan administered in these methods.

Delivery of the formulation can be continuous over a pre-selectedadministration period ranging from several hours, one to several weeks,one to several months, or up to one or more years. In certainembodiments, the dosage form is one that is adapted for delivery of GAAover an extended period of time. Such delivery devices may be adaptedfor administration of GAA for several hours (e.g., 2 hours, 12 hours, or24 hours to 48 hours or more), to several days (e.g., 2 to 5 days ormore, from about 100 days or more), to several months or years. In someof these embodiments, the device is adapted for delivery for a periodranging from about 1 month to about 12 months or more. The GAA deliverydevice may be one that is adapted to administer GAA to an individual fora period of, e.g., from about 2 hours to about 72 hours, from about 4hours to about 36 hours, from about 12 hours to about 24 hours; fromabout 2 days to about 30 days, from about 5 days to about 20 days, fromabout 7 days to about 100 days or more, from about 10 days to about 50days; from about 1 week to about 4 weeks; from about 1 month to about 24months or more, from about 2 months to about 12 months, from about 3months to about 9 months; or other ranges of time, including incrementalranges, within these ranges, as needed.

In certain embodiments, the methods of the invention includeadministering to an individual, for example, subcutaneousadministration, a dose of from about 0.1 to about 50 mg/kg of GAA,wherein the dose is administered once per day, once every two days, onceevery three days, once every four days, once every five days, or onceevery six days. In certain embodiments, the formulation of the presentapplication is administered once per week, twice per week, three timesper week, four times per week, five times per week, six times per weekor seven times per week.

In certain embodiments, the methods of the present application compriseadministering a co-formulation to an individual comprising GAA and anASSC, wherein the co-formulations is administered subcutaneously. Incertain embodiments, the dose of GAA in the co-formulation is betweenabout 0.1 and about 5000 mg/kg, or between about 10 and about 4000mg/kg, or between about 25 and about 3000 mg/kg, or between about 50 andabout 2000 mg/kg, or between about 100 and about 1000 mg/kg, or betweenabout 200 and about 500 mg/kg.

In certain embodiments, the dose of GAA in the co-formulation is betweenabout 0.1 and about 100 mg/kg, or between about 1 and about 80 mg/kg, orbetween about 5 and about 50 mg/kg, or between about 10 and about 40mg/kg, or between about 15 and about 25 mg/kg.

In certain embodiments, the dose of rhGAA in the co-formulation is about20 mg/kg.

In certain embodiments, the dose of ASSC in the co-formulation isbetween about 0.1 and about 5000 mg/kg, or between about 10 and about4000 mg/kg, or between about 25 and about 3000 mg/kg, or between about50 and about 2000 mg/kg, or between about 100 and about 1000 mg/kg, orbetween about 200 and about 500 mg/kg.

In certain embodiments, the dose of ASSC in the co-formulation isbetween about 0.1 and about 100 mg/kg, or between about 1 and about 80mg/kg, or between about 5 and about 50 mg/kg, or between about 10 andabout 40 mg/kg, or between about 15 and about 25 mg/kg.

In certain embodiments, the dose of ASSC in the co-formulation is about30 mg/kg.

In certain embodiments, the dose does not result in a toxic level of GAAin the liver of the individual. In some embodiments, the GAA isadministered in a sufficient dose to result in a peak concentration ofGAA in tissues of the subject, for example muscle tissue, within about24 hours after the administration of the dose. In certain embodiments,the GAA is administered in a sufficient dose to result in a peakconcentration of GAA in tissues of the subject within about 10 to about50 hours, or about 45, 40, 35, 30, 25, or fewer hours after theadministration of the dose. In some embodiments, the formulation of theGAA is a single-dose formulation. In some embodiments, the formulationof the GAA is a multi-dose formulation.

A GAA preparation of the present invention can be formulated such thatthe total required dose may be administered in a single subcutaneousinjection of one or more milliliters, for example, 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more milliliters. The preparation can be formulated to beadministered subcutaneously at several different injection sites. Inorder to allow an injection volume of one or two milliliters, a GAApreparation of the present invention may be formulated at aconcentration in which the preferred dose is delivered in a volume ofone to two milliliters. Subcutaneous injections of GAA preparations havethe advantages of being convenient for the patient, in particular byallowing self-administration, while also resulting in a prolonged plasmahalf-life as compared to, for example, intravenous administration. Aprolongation in plasma half-life results in maintenance of effectiveplasma GAA levels over longer time periods, the benefit of which is toincrease the exposure of clinically affected tissues to the injected GAAand, as a result, may increase the uptake of GAA into such tissues. Thisallows a more beneficial effect to the patient and/or a reduction in thefrequency of administration. Furthermore, a variety of devices designedfor patient convenience, such as refillable injection pens andneedle-less injection devices, may be used with the GAA preparations ofthe present invention as discussed herein. Because the GAA co-formulatedwith an ASSC is stable at room temperature for extended periods of time,the preparation can be held in a cartridge aside the patient's body toallow for recurring low dose administration or continuous low volumeadministration to provide for a steady state of enzyme administration.Such administration may avoid the potential side effects of high dosesof enzyme replacement therapy (ERT) administered during an intravenousinfusion.

5.5 Pharmaceutical Compositions

The compounds and compositions of the invention may be formulated aspharmaceutical compositions by admixture with a pharmaceuticallyacceptable carrier or excipient.

In one embodiment, an ASSC and GAA are formulated in a singlecomposition (i.e., a co-formulation). Such a composition enhancesstability of GAA both during storage (i.e., in vitro) and in vivo afteradministration to a subject, thereby increasing circulating half-life,tissue uptake, and resulting in increased therapeutic efficacy of GAA.The formulation is preferably suitable for parenteral administration,including intravenous, subcutaneous, and intraperitoneal administration,however, formulations suitable for other routes of administration suchas oral, intranasal, or transdermal are also contemplated.

The present invention features liquid pharmaceutical formulations (e.g.,formulations comprising GAA and an ASSC) having improved properties ascompared to art-recognized formulations. The present invention is basedon the surprising finding that by combining an ASSC with GAA, theconcentration of GAA in a formulation can be increased to an amount thatwould normally result in the formation of GAA aggregates in the absenceof an ASSC. Despite the high concentration of GAA, the formulation ofthe invention is able to maintain solubility and stability of the GAA,e.g., during manufacturing, storage, and/or repeated freeze/thawprocessing steps or extended exposure to increased air-liquidinterfaces. In addition, the formulation of the invention maintains alow level of protein aggregation (e.g., less than about 5%, 4%, 3%, 2%,or less than about 1%), despite having a high concentration of GAA. Theformulation of the invention also surprisingly maintains a low viscositywithin ranges suitable for subcutaneous injection, despite having a highconcentration of GAA.

The present invention also provides for very potent and concentrated GAAformulations that can be achieved by solubilizing the GAA in a smallvolume by combining the GAA with an ASSC. The formulations of theinvention are of particular use where the delivery device is relativelysmall (e.g., an implantable system), where delivery is required for arelatively long duration, or where high effective doses of GAA arerequired to achieve the desired therapeutic effect. Thus, it is possibleto deliver a consistent amount of GAA over an extended period of time(e.g., days, weeks, months, etc.) without the need to refill or replacethe delivery device, thereby reducing risk of infection and tissuedamage, increasing patient compliance, and achieving consistent,accurate dosing.

In certain embodiments of the present invention, therapeutic amounts ofGAA (even high doses) can be delivered to a subject by using only verysmall volumes of GAA (e.g., on the order of microliters per day ornanoliters per day). In certain body tissues, e.g., subcutaneous space,low volume delivery facilitates better absorption of the GAA by thelocal tissue, and minimizes local tissue disturbance, trauma, or edema.

In certain embodiments, formulations of the invention include highconcentrations of GAA such that the liquid formulation does not showsignificant opalescence, aggregation, or precipitation.

In another embodiment, formulations of the invention include highconcentrations of GAA such that are suitable for, e.g., subcutaneousadministration without significant felt pain (e.g., as determined by avisual analog scale (VAS) score).

In certain embodiments, the formulations of the invention comprise ahigh GAA concentration, including, for example, a GAA concentration ofabout 25 mg/mL, or about 50 mg/mL, or about 80 mg/mL, or about 100mg/mL, or about 115 mg/mL, or about 150 mg/ml, or about 160 mg/ml orabout 200 mg/mL, or about 240 mg/mL, or about 250 mg/mL. For example, asdescribed in Example 10 below, in one aspect of the invention the liquidpharmaceutical formulation comprises a human recombinant wild-type GAAconcentration of about 25 mg/mL. It is also contemplated that theformulations of the invention may comprise a GAA concentration betweenabout 1 mg/mL and about 500 mg/mL, or between about 5 mg/mL and about500 mg/mL, or between about 5 mg/mL and about 250 mg/mL, or betweenabout 10 mg/mL and about 200 mg/mL, or between about 20 mg/mL and about100 mg/mL, or between about 1 mg/mL and about 60 mg/mL. Concentrationsand ranges intermediate to the above recited concentrations are alsointended to be part of this invention (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, or 200 mg/mL).

In certain embodiments, a formulation of the invention comprises a GAAin a concentration that is greater then 5 mg/mL.

In certain embodiments, a formulation of the invention comprises an ASSCin an amount effective to reduce or inhibit aggregation of GAA in theformulation. Such an amount of ASSC includes, for example, between about0.005 mM and 100 mM, or between about 0.05 mM and about 90 mM, orbetween about 0.1 mM and about 80 mM, or between about 0.5 mM and about70 mM, or between about 1 mM and about 60 mM, or between about 2 mM andabout 50 mM, or between about 3 mM and about 40 mM, or between about 4mM and about 30 mM, or between about 5 mM and about 20 mM. In certainembodiments, the ASSC is present in the formulations of the invention ata concentration of between about 0.5 and about 20 mM. In certainembodiments, the ASSC is present in the formulations of the invention ata concentration of about 1 mM. In certain embodiments, the ASSC ispresent in the formulations of the invention at a concentration of about10 mM.

In certain embodiments, a formulation of the invention comprises an ASSCin an amount effective to reduce or inhibit aggregation of GAA in theformulation. Such an amount of ASSC includes, for example, between about5 and about 500 mg/mL, or between about 10 and about 250 mg/mL, orbetween about 20 and about 200 mg/mL, or between about 30 and about 150mg/mL, or between about 40 and about 100 mg/mL, or between about 50 andabout 75 mg/mL. In certain embodiments, the ASSC is present in an amountof between about 5 and about 200 mg/mL.

In certain embodiments, the ASSC is present in the formulation at aconcentration of about 32 mg/mL or about 160 mg/mL.

In certain embodiments of the invention, a liquid formulation comprisingDNJ and GAA is prepared by dissolving DNJ in water to achieve aconcentration of 10 mM DNJ. GAA can be reconstituted in 1.8 ml water,and dialyzed overnight in phosphate buffered-saline (pH 7.4). 4.4microliters of DNJ (10 mM) can then be added to 400 microliters of GAA,such that the GAA is at a concentration of 25 mg/ml.

In another embodiment, the GAA and the ASSC are formulated in separatecompositions. In this embodiment, the chaperone and the replacementprotein may be administered according to the same route, e.g.,intravenous infusion, or different routes, e.g., intravenous infusionfor the replacement protein, and oral administration for the ASSC.

The pharmaceutical formulations suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, benzyl alcohol, sorbic acid,and the like.

In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonosterate and gelatin. Sterile injectable solutions may be prepared byincorporating the GAA and ASSC in the required amounts in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filter or terminal sterilization.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze-drying technique which yield a powder ofthe active ingredient plus any additional desired ingredient frompreviously sterile-filtered solution thereof.

Preferably the formulation may contain one or more excipients.Pharmaceutically acceptable excipients which may be included in theformulation are buffers such as citrate buffer, phosphate buffer (suchas, for example, monobasic sodium phosphate, dibasic sodium phosphateand combinations thereof), acetate buffer, bicarbonate buffer, aminoacids, urea, alcohols, ascorbic acid, phospholipids; proteins, such asserum albumin, collagen, and gelatin; salts such as EDTA or EGTA, andsodium chloride; liposomes; polyvinylpyrollidone; sugars, such asdextran, mannitol, sorbitol, and glycerol; propylene glycol andpolyethylene glycol (e.g., PEG-4000, PEG-6000); glycerol; glycine orother amino acids; and lipids. Buffer systems for use with theformulations include citrate; acetate; bicarbonate; and phosphatebuffers.

In certain embodiments, the formulations of the present applicationfurther comprise an excipient selected from the group consisting ofpolyethylene glycol (PEG), PEG-400, arginine, arginine and glutamicacid, proline, gamma-cyclodextrin and combinations thereof.

In certain embodiments, the buffer and/or excipient is present in theformulation at a concentration of between about 1 and about 50%weight/volume (w/v), or between about 2 and about 40% w/v, or betweenabout 3 and about 30% w/v, or between about 4 and about 20% w/v, orbetween about 5 and about 10% w/v.

In certain embodiments, the buffer and/or excipient is present in theformulation at a concentration of between about 1 and about 500 mM, orbetween about 10 and about 400 mM, or between about 20 and about 300 mM,or between about 30 and about 250 mM, or between about 40 and about 200mM, or between about 50 and about 150 mm, or between about 60 and about100 mM.

In certain embodiments, the formulation comprises phosphate bufferpresent at a concentration of about 26 mM.

In certain embodiments, the formulation comprises citrate buffer presentat a concentration of about 150 mM.

In certain embodiments, the formulation comprises PEG-400 present at aconcentration of about 5% w/v.

In certain embodiments, the formulation comprises arginine present at aconcentration of about 100 mM.

In certain embodiments, the formulation comprises arginine at aconcentration of about 50 mM and glutamic acid present at aconcentration of about 50 mM.

In certain embodiments, the formulation comprises proline present at aconcentration of about 250 mM.

In certain embodiments, the formulation comprises gamma-cyclodextrinpresent at a concentration of about 10% w/v.

The formulation also may contain a non-ionic detergent. Preferrednon-ionic detergents include Polysorbate 20, Polysorbate 80, TritonX-100, Triton X-114, Nonidet P-40, Octyl α-glucoside, Octyl f-glucoside,Brij 35, Pluronic, and Tween 20.

For lyophilization of protein and chaperone preparations, the proteinconcentration can be 0.1-10 mg/mL. Bulking agents, such as glycine,mannitol, albumin, and dextran, can be added to the lyophilizationmixture. In addition, possible cryoprotectants, such as disaccharides,amino acids, and PEG, can be added to the lyophilization mixture. Any ofthe buffers, excipients, and detergents listed above, can also be added.

The route of administration may be oral or parenteral, includingintravenous, subcutaneous, intra-arterial, intraperitoneal, ophthalmic,intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral,intradermal, intracranial, intraspinal, intraventricular, intrathecal,intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal,transdermal, or via inhalation.

Administration of the above-described parenteral formulations may be byperiodic injections of a bolus of the preparation, or may beadministered by intravenous or intraperitoneal administration from areservoir which is external (e.g., an i.v. bag) or internal (e.g., abioerodable implant, a bioartificial organ, or a population of implantedcells that produce the replacement protein). See, e.g., U.S. Pat. Nos.4,407,957 and 5,798,113, each incorporated herein by reference.Intrapulmonary delivery methods and apparatus are described, forexample, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aeorosolizer, electroporation, and transdermalpatch. Needle-less injector devices are described in U.S. Pat. Nos.5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the formulationsdescribed above can administered in these methods.

5.6 In Vitro Stability

Ensuring the stability of GAA formulations during its shelf life is amajor challenge. For example, the patient instructions for Myozyme® andLumizyme® notes that vials are for single use only and that unusedproduct should be discarded. The instructions further state thatMyozyme® and Lumizyme® should be reconstituted, diluted, andadministered by a health care professional, and that administrationshould be without delay. Myozyme® and Lumizyme® must be stored at 2 to8° C., and the product is only stable for up to 24 hours at thesetemperatures.

When an ASSC and the GAA are present in the same composition, theformulated compositions of the invention provide more stablecompositions. In addition to stabilizing the administered protein invivo, the ASSC reversibly binds to and stabilizes the conformation ofthe GAA in vitro, thereby preventing aggregation and degradation, andextending the shelf-life of the formulation. Analysis of theASSC/replacement protein interaction may be evaluated using techniqueswell-known in the art, such as, for example, differential scanningcalorimetry, or circular dichroism.

For example, where an aqueous injectable formulation of the compositionis supplied in a stoppered vial suitable for withdrawal of the contentsusing a needle and syringe, the presence of an ASSC inhibits aggregationof the GAA. The vial could be for either single use or multiple uses.The formulation can also be supplied as a prefilled syringe, anautoinjector pen, or a needle-free administration device. In anotherembodiment, the formulation is in a dry or lyophilized state, whichwould require reconstitution with a standard or a supplied,physiological diluent to a liquid state. In this instance, the presenceof an ASSC stabilizes the GAA during and post-reconstitution to preventaggregation. In the embodiment where the formulation is a liquid forintravenous administration, such as in a sterile bag for connection toan intravenous administration line or catheter, the presence of an ASSCconfers the same benefit.

In addition to stabilizing the replacement protein to be administered,the presence of an ASSC enables the GAA formulation to be stored at aneutral pH of about 7.0-7.5. This confers a benefit to proteins thatnormally must be stored at a lower pH to preserve stability. Forexample, lysosomal enzymes, such as GAA, typically retain a stableconformation at a low pH (e.g., 5.0 or lower). However, extended storageof the replacement enzyme at a low pH may expedite degradation of theenzyme and/or formulation.

As described above, the liquid formulation of the invention hasadvantageous stability and storage properties. Stability of the liquidformulation is not dependent on the form of storage, and includes, butis not limited to, formulations which are frozen, lyophilized,spray-dried, or formulations which in which the active ingredient issuspended. Stability can be measured at a selected temperature for aselected time period. In one aspect of the invention, the protein in theliquid formulations is stable in a liquid form for at least about 1week; at least about 2 weeks; at least about 3 weeks; at least about 1month; at least about 2 months; at least about 3 months; at least about4 months, at least about 5 months; at least about 6 months; at leastabout 12 months; at least about 18 months. Values and rangesintermediate to the above recited time periods are also intended to bepart of this invention, e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 months. In addition,ranges of values using a combination of any of the above recited valuesas upper and/or lower limits are intended to be included. In certainembodiments, the formulation is stable at room temperature (about 30°C.) or at about 37° C., or at about 40° C., or at about 45° C. for atleast about 1 month and/or stable at about 2-8° C. for at least about 1year, or more preferably stable at about 2-8° C. for at least about 2years. Furthermore, the formulation is preferably stable followingfreezing (to, e.g.,−80° C.) and thawing of the formulation, hereinafterreferred to as a “freeze/thaw cycle.”

Stability of a protein (e.g., protein stability and/or reduction incontamination) in a liquid formulation may also be defined as thepercentage of monomer, aggregate, or fragment, or combinations thereof,of the protein in the formulation. A protein “retains its physicalstability” in a formulation if it shows substantially no signs ofaggregation, precipitation and/or denaturation upon visual examinationof color and/or clarity, or as measured by UV light scattering or bysize exclusion chromatography, non-denaturing PAGE, or other methods fordetermining size, etc. In one aspect of the invention, a stable liquidformulation is a formulation having less than about 10%, or less thanabout 5%, or less than about 1% of the protein being present asaggregate in the formulation.

In one embodiment, the physical stability of a liquid formulation isdetermined by determining turbidity of the formulation following a stirstress assay, e.g., 24 hour or 48-hour stir-stress assay. For example, astir stress assay may be performed by placing a suitable volume of aliquid formulation in a beaker with a magnetic stirrer, e.g.,(multipoint HP, 550 rpm), removing aliquots at any suitable time, e.g.,at T0-T48 (hrs), and performing suitable assays as desired on thealiquots. Samples of a formulation under the same conditions but withoutstirring serve as control.

Turbidity measurements may be performed using a laboratory turbiditymeasurement system from Hach (Germany) and are reported as nephelometricunits (NTU).

Stability of the composition (e.g., protein stability and/or reductionin contamination) can also be measured, e.g., by measuring proteindegradation or contaminant growth or presence. Protein degradation canbe determined, e.g., by reverse phase HPLC, non-denaturing PAGE,ion-exchange chromatography, peptide mapping, or similar methods.

The stability of GAA in the presence of an ASSC, at a concentrationdescribed herein, can be measured, e.g., as a percent aggregation ordegradation, at a predetermined time and compared with one or morestandards. For example, a suitable standard is a composition similar tothe test conditions except that the GAA is not contacted with an ASSC.The stabilities of the GAA at a concentration are compared. Suitabilitycan be shown by the GAA at a particular concentration in combinationwith an ASSC having comparable or better stability than in the absenceof the ASSC.

5.7 In Vivo Stability

As described above for the in vitro formulations, the presence of anASSC for the GAA has the benefit of prolonging in plasma the half-lifeof the exogenous GAA, thereby maintaining effective replacement proteinlevels over longer time periods, resulting in increased exposure ofclinically affected tissues to the GAA and, thus, increased uptake ofprotein into the tissues. This confers such beneficial effects to thepatient as enhanced relief, reduction in the frequency, and/or reductionin the amount administered. This will also reduce the cost of treatment.

In addition to stabilizing wild-type replacement GAA, the ASSC will alsostabilize and enhance expression of endogenous mutant GAA that aredeficient as a result of mutations that prevent proper folding andprocessing in the ER, as in conformational disorders such as PompeDisease.

The present invention is not to be limited in scope by the specificembodiments described herein and the Examples that follow. Indeed,various modifications of the invention in addition to those describedherein will become apparent to those skilled in the art from theforegoing description and the accompanying Examples and Figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

EXAMPLES Example 1: In Vitro Thermal Stability of rhGAA and 100 μM1-DNJ-HCl

The stability of recombinant human GAA (Myozyme®, Genzyme Corp.) withand without 100 μM of the ASSC 1-deoxynojirimycin hydrochloride(1-DNJ-HCl) was determined via a thermal stability assay that utilizesheat to induce protein denaturation. Denaturation is monitored using aSYPRO Orange dye that fluoresces upon binding to hydrophobic amino acids(which are not exposed in a folded protein).

The thermal stability was performed at pH 7.4 for two formulations,which corresponds to the pH of the ER. As shown in FIG. 1, theformulation that contains 100 μM of 1-DNJ-HCl at 7.4 pH requiredsignificantly more heat to denature, and is thus more stable, ascompared to formulation without the ASSC at 7.4 pH.

Example 2: GAA Residual Activity and Thermal Stability of rhGAA and 50μM 1-DNJ-HCl

Residual GAA activity was determined for four formulations:

-   -   (1) Myozyme® alone at pH 7.4;    -   (2) Myozyme® plus 50 μM 1-DNJ-HCl at pH 7.4;    -   (3) Myozyme® alone at pH 5.2;    -   (4) Myozyme® plus 50 μM 1-DNJ-HCl at pH 5.2.

Activity was measured, based on the % of initial activity (t=0) over 24hours. Samples were assayed for GAA enzyme activity based on thehydrolysis of the fluorogenic substrate 4-MU-α-glucose at 0, 3, 6 and 24hours. The GAA activity was expressed as % of initial activity, i.e.residual activity.

As shown in FIG. 2A, formulation (1) above (without the ASSC) lostactivity over time, having only about 20% of its initial activity 24hours after administration. In contrast, formulation (2) maintainedmost, if not all of its initial activity over 24 hours. Bothformulations at ph 5.2 (formulations (3) and (4) above) maintained mostof their initial activity over 24 hours.

In order to determine if loss of initial enzyme activity is correlatedto failure to maintain a proper conformation, a SYPRO Orange thermalstability experiment was performed on the samples above as generallydescribed in Example 1. In this thermal stability experiment, however,the concentration of 1-DNJ-HCl was increased to 100 μM in formulations(2) and (4). Based on this experiment, the % of GAA folded was estimatedand plotted in FIG. 2B. The decrease in the amount of folded GAA over 24hours in FIG. 2B for the formulation (1) correlates to the loss ofactivity shown in FIG. 2A for this same general formulation.

Example 3: In Vivo Uptake of Myozyme® in GAA KO Mice with and withoutOral Administration of 1-DNJ-HCl

Five groups of GAA KO mice were administered one of the followingformulations:

-   -   (1) untreated control;    -   (2) 10 mg/kg of Myozyme® IV once per week, for up to three weeks    -   (3) Myozyme® infusion as in (2), plus 10 mg/kg of 1-DNJ-HCl;    -   (4) Myozyme® infusion as in (2), plus 100 mg/kg of 1-DNJ-HCl;    -   (5) Myozyme® infusion as in (2), plus 1000 mg/kg of 1-DNJ-HCl;        Tissue homogenates were generated for analysis. Enzymatic        activity as determined using a 4-MUG fluorogenic substrate        assay. The results are shown in FIG. 3.

These results indicate that Myozyme® tissue uptake (as a measure of GAAactivity) declined at 7 days post injection for all groups.Coadministration of 1-DNJ-HCl with Myozyme® facilitated a dose-dependentincrease in Myozyme® uptake for up to 7 days post injection. The effectof 1-DNJ-HCl was more pronounced and significant (p<0.05 t-test vs.Myozyme® alone) at 4 and 7 days post injection of either 1, 2, or 3doses.

Example 4: In Vivo Uptake of Myozyme® in GAA KO Mice with and withoutOral Administration of 1-DNJ-HCl

A thermal stability experiment as generally described in Example 1 wasperformed on four compositions:

-   -   (1) Myozyme® only composition;    -   (2) Myozyme® plus 1 μM of 1-DNJ-HCl;    -   (3) Myozyme® plus 10 μM of 1-DNJ-HCl;    -   (4) Myozyme® plus 100 μM of 1-DNJ-HCl;        As shown in FIG. 5, DNJ-HCl increases GAA thermostability as        evident by increases in GAA's melting temperature in a        dose-dependent manner.

Example 5: In Vivo Half-Life of rhGAA In Rats When Administered asMonotherapy, or when Combined with 1-DNJ-HCl

Four groups of rats were administered one of the following dosingregimens:

-   -   (1) Saline+Water;    -   (2) 10 mg/kg of rhGAA+Water;    -   (3) 10 mg/kg of rhGAA+3 mg/kg of 1 DNJ-HCl;    -   (4) 10 mg/kg of rhGAA+30 mg/kg of 1 DNJ-HCl;

The rhGAA or saline was administered 30 minutes after administration ofthe 1-DNJ-HCl. GAA Activity was determined as generally described inExample 3. The results over 24 hours are shown in FIG. 6. The 1-DNJ-HClinhibited the loss of enzyme activity post-administration, therebyincreasing the in vivo half life of rhGAA. The in vivo half life ofrhGAA increased from 1.4±0.2 hours (0 mg/kg of 1-DNJ-HCl) to 2.1±0.2hours (3 mg/kg of 1-DNJ-HCl) and 3.0±0.4 hours (30 mg/kg of 1-DNJ-HCl).

Example 6: GAA Enzyme Activity in GAA KO Mouse

Three groups of GAA KO Mice were administered one of the followingformulations:

-   -   (1) Control (No Treatment);    -   (2) 10 mg/kg of rhGAA;    -   (3) 10 mg/kg of rhGAA and 100 mg/kg of 1-DNJ-HCl 30 minutes        prior to rhGAA infusion, and every 8 hours after infusion for 48        hours.

Heart and Diaphragm tissue homogenates were harvested and rhGAA activitywas measured using the fluorogenic substrate (4-MUG). The results areshown in FIGS. 7A and 7B.

Example 7: 1-DNJ-HCl Stabilizes rhGAA and Prevents Enzyme Inactivationin Blood

1-DNJ-HCl was evaluated for its ability to stabilize rhGAA (e.g.,Myozyme®) in whole (sodium citrate anti-coagulated) blood at 37° C. tomimic the environment that the ERT is exposed to during the multi-hourinfusion. The results indicate that rhGAA is unstable under theseconditions such that approximately 40% of the enzyme inactivated by 4hrs, ˜70% by 8 hrs and nearly 100% by 24 hrs as shown (red diamond lineplot) in FIG. 8. These results suggest that a significant fraction ofthe rhGAA dose would likely be inactive because these infusions aretypically more than 6 hrs, and in some instances 12 hrs. Moreover, sinceMyozyme® has a long plasma half-life (reported to be more than 3 hrs),there is a high probability that an appreciable amount of the enzymeremains in the circulation many hours after the infusion that would alsobe prone to inactivation. By contrast, when rhGAA was incubated with 50μM 1-DNJ-HCl under the same experimental conditions, the enzyme remainedcompletely active throughout the study (blue square line plot). Theseresults indicate that 1-DNJ-HCl stabilized rhGAA and prevented enzymeinactivation in whole blood. Importantly, these data also indicate thatthe plasma proteins present in blood are not sufficient to prevent theloss of rhGAA enzyme activity whereas a pharmacological chaperone like1-DNJ-HCl is able to prevent enzyme inactivation.

Example 8: 1-DNJ-HCl Stabilizes rhGAA and Prevents Enzyme Inactivationin Blood

rhGAA measured in whole blood with varying concentrations of 1-DNJ-HCl(0-100 μM) to determine the minimum concentration of 1-DNJ-HCl thatprevents rhGAA enzyme inactivation (FIG. 9). As expected, high 1-DNJ-HClconcentrations (50 and 100 μM) were best for stabilizing rhGAA andpreventing to enzyme inactivation. Interesting however, low 1-DNJ-HClconcentrations (as low as 2.5 μM) also maintained rhGAA activity with aloss of ˜20% over a 6-hr time course. These results suggest thatmoderate 1-DNJ-HCl concentrations (e.g., 10-25 μM) may be adequate forstabilizing rhGAA in blood during infusions. Based on human plasma PKdata, these concentrations are readily obtainable in the clinic.

Example 9: Myozyme® Co-Administered with 1-DNJ-HCl Resulted inSignificantly Greater Tissue Glycogen Reduction in GAA KO Mice asCompared to Myozyme® Alone

Twelve-week old male GAA KO mice were administered a single dose ofMyozyme® (40 mg/kg) via bolus tail vein injection every other week for 8weeks. To prevent anaphylaxis, before the third and fourth Myozyme®injection, diphenhydramine (10 mg/kg intraperitoneally) was administered10 min before Myozyme® injection. In addition, mice received eitherwater or 30 mg/kg of 1-DNJ-HCl administered via oral gavage 30 minutesprior to Myozyme® administration. Mice were euthanized 14 days after thelast Myozyme® administration. The Experimental design is shown in FIG.10.

Glycogen levels in heart, diaphragm, soleus, and quadriceps were thenmeasured. Myozyme® co-administered with 1-DNJ-HCl resulted insignificantly greater tissue glycogen reduction in GAA KO mice ascompared to Myozyme® alone (FIGS. 11A, 11B, 11C, and 11D). Briefly,homogenates were prepared by homogenizing ˜50 mg tissue for 3-5 secondson ice with a microhomogenizer in 200 μL deionized water. Supematantswere heat denatured (99° C. for 10 min) to remove endogenousamyloglucosidase activity. Denatured lysates (4 μL) were then analyzedin duplicate by addition of 36 μL water with and without 10 μL of 800U/mL of amyloglucosidase (Sigma Aldrich, St. Louis, Mo.) and incubatedfor 1 hour at 50° C. The reaction was stopped by inactivation at 100° C.for 10 min. Finally, 200 μL of glucose reagent (Sigma) was addedabsorbance read at 340 nm on Spectramax. A standard curve ranging from 5μg/mL to 400 μg/mL Type III rabbit liver glycogen (Sigma) was run eachday for conversion of absorbance to absolute glycogen units.Simultaneously, the amount of protein was determined in tissuehomogenates using the Micro BCA Protein Assay (Pierce, Rockford, Ill.)following the manufacturer's instructions. The glycogen content of eachsample was normalized to protein, and data were finally expressed asmicrograms of glycogen per milligram of protein (μg/mg protein).

Example 10: DNJ Reduces Aggregation of Myozyme® in CompositionsComprising High Concentrations of Myozyme®

A liquid formulation comprising DNJ and GAA was prepared by dissolvingDNJ in water to achieve a concentration of 10 mM DNJ. GAA wasreconstituted in 1.8 ml water, and dialyzed overnight in phosphatebuffered-saline (pH 7.4). 4.4 microliters of DNJ (10 mM) was then beadded to 400 microliters of GAA, such that the GAA was at aconcentration of 25 mg/ml.

25 mg/mL Myozyme® was incubated with or without 1 mM DNJ inphosphate-buffered saline at pH 7.4. Aggregation of Myozyme® wasassessed after incubation for 4 weeks at 37° C. As shown in FIG. 12,combining 1 mM DNJ with 25 mg/mL Myozyme® reduced aggregation ofMyozyme®.

Example 11: DNJ Increases the Circulating Half-Life and Tissue Uptake ofMyozyme®

Sprague-Dawley rats were administered 10 mg/kg Myozyme® or 30 mg/kg DNJmixed with 10 mg/kg Myozyme® via tail vein. GAA activity was measured inplasma and quadriceps tissue. Baseline GAA activity in quadriceps wassubtracted (˜16 nmol/mg protein/hr) from the measured GAA following GAAor DNJ and GAA administration.

As shown in FIG. 13, administering 30 mg/kg DNJ with 10 mg/kg Myozyme®via tail vein increased the circulating plasma half-life and tissueuptake of Myozyme® in quadriceps.

Example 12: Solubility of Lumizyme® in the Presence of 1-DNJ

The solubility of Lumizyme® (alglucosidase alpha) in the presence of1-DNJ and different excipients was examined.

Method

A vial of Lumizyme® containing 52.5 mg protein, 210 mg mannitol, 0.5 mgpolysorbate 80, 9.9 mg Na₂HPO₄×7H₂O, and 31.2 mg NaH₂PO₄×H₂O wasdissolved in a minimal volume of water (1 mL added) yielding 1.2 mL ofprotein solution. Five 240-μL aliquots (each containing 10.5 mg protein)were transferred to Amicon Ultra 0.5 mL 30 kDa cutoff centrifugal filterdevices and centrifuged for 10 min at 14,000×g in an Eppendorfcentrifuge to obtain about 50 μL of retentate.

The original excipients present in the composition, i.e., mannitol andpolysorbate 80 were exchanged for new excipients to test proteinsolubility (keeping the same buffer conditions). The following excipientsolutions were prepared for the exchange using the original phosphatebuffer:

-   -   1. PEG 400 (5% w/v)    -   2. Arginine (100 mM)    -   3. Arginine (50 mM)+Glutamic acid (50 mM)    -   4. Proline (250 mM)    -   5. Gamma-cyclodextrin (10%)        All samples contained the small molecule ligand        1-deoxynojirimycin hydrochloride (1-DNJ-HCl, AT2220 HCl, 2220        HCl) at a ratio 1:1 (w/w) of the ligand to protein, except for        sample 2 (100 mM arginine) which contained the ligand at a        reduced 1:5 (w/w) ligand to protein ratio.

For exchanging excipients, 5.2 mg Na₂HPO₄ and 27.1 mg NaH₂PO₄ weredissolved in 10.3 mL water to prepare the washing/excipient exchangephosphate buffer of approximately 26 mM. A volume of 8 mL of the bufferfor excipient exchange was supplemented with 160 mg/mL of the smallmolecule ligand (to prepare excipient 1, 3, 4 and 5 solutions), while 2mL was supplemented with 32 mg/mL ligand for excipient 2 (100 mMArginine). Each of the excipients were dissolved in 2 mL of this bufferat the concentrations listed above.

An excipient solution was added to each separate filter device toachieve a total volume of 0.5 mL. Each filter device was centrifuged for15 min at 14,000×g in a bench top Eppendorf centrifuge to retainapproximately 25 μL. The units were re-filled with the same excipientsolution to 0.5 mL and centrifuged again (this process was repeated 2times). This procedure efficiently replaced mannitol and polysorbate 80with excipients described above, diluting the original constituents byabout 1:1000. All filtrates for each excipient were collected andexamined for any protein leaks through the filter.

Each centrifugal filter device was inverted in a clean tube, and theconcentrated protein solution was collected from each filter device bycentrifugation for 2 min at 1000×g. The volume of each sample wasdetermined (66 μL, corresponding to a target protein concentration of160 mg/mL), and it was confirmed that no visible precipitate wasobservable in each sample. Samples were transferred back to theirrespective filter devices and centrifuged for an additional 5 min.Samples were then collected by inversion of the filters into clean tubesand centrifuging for 2 min at 1000×g to yield about 37 μL of retentate(corresponding to >280 mg/mL protein based on a total initialconcentration of 10.5 mg protein).

The samples were vortexed and incubated for 1 hr at room temperature toequilibrate liquid and solid phases followed by centrifugation for 10min at 14,000×g. Again, no visible precipitate was observed. Two-three10 μL aliquots were taken from the supernatant of each sample (dependingon available volume of the liquid phase), and diluted 1:1000 in twosteps. Protein concentration was measured by UV absorbance at 280 nm,and the maximum protein solubility was calculated according to a highlyreproducible calibration curve. The calibration curve was prepared bydissolving 6.8 mg of Myozyme® (alglucosidase alpha) lyophilized powder(containing 1.16 mg protein) in 1.16 mL of water followed by a serialdilution in deionized water. Absorbance was measured at 280 nm in a 1 cmlight-pass quartz semi-micro cuvette using 0.5 mL aliquots.

Results:

As shown in Table 1, the excipients tested in this study increased thesolubility of the protein in the presence of a small molecule ligand,1-DNJ-HCl. The highest value observed was for the mixture of arginineand glutamic acid (242 mg/mL), the lowest for gamma-cyclodextrin (114mg/mL). The solubility of the protein without the added excipients wasdetermined to be about 80 mg/mL. Without being bound by any theory, theincrease in solubility may be due to efficient interactions of aminoacid excipients with the protein surface hydrophobic and hydrophilicsites, which competitively block protein-protein associations.

TABLE 1 Solubility of Lumizyme ® (alglucosidase alpha) in the presenceof the ligand 1-DNJ-HCl and selected excipients. Protein 1-DNJ-HClsolubility Excipient Concentration (mg/mL) (mg/mL) PEG400 5% (w/v) 160158 Arginine 100 mM 32 201 Arginine + Glutamic acid 50 mM + 50 mM 160242 Proline 250 mM 160 195 Gamma-cyclodextrin 10% (w/v) 160 114

Example 13: The Effect of Repeat Subcutaneous Dosing of Co-FormulatedrhGAA and 1-DNJ on Tissue Uptake and Glycogen Reduction in GAA KO Mice

The present study examined whether GAA knockout mice (GAA KO) toleraterepeat subcutaneous (SQ) injections of rhGAA or a co-formulation ofrhGAA and 1-DNJ, whether the repeated SQ injections of rhGAA increasetissue uptake of rhGAA and reduce glycogen levels in the GAA KO mice,and whether the repeated SQ injections of a co-formulation of rhGAA and1-DNJ increase tissue uptake of rhGAA and reduce glycogen levelscompared to SQ injections of rhGAA alone.

Methods

12-week old male GAA KO mice in groups of 7 were used in the presentstudy. Each group of mice was administered one of the followingtreatments:

-   -   (1) Saline only (no drug control);    -   (2) Lumizyme® alone (20 mg/kg) delivered subcutaneously (SQ); or    -   (3) Co-formulated Lumizyme® (20 mg/kg) and 1-DNJ (30 mg/kg) SQ.

Each of the treatments were administered to their respective treatmentgroup for a period of 2 weeks. The treatments were administered onMonday and Thursday of each week. SQ injections were administeredbetween the shoulder blades of each mouse receiving treatment. A totalof 4 doses was administered to each study animal. Diphenhydramine wasadministered intraperitoneally (IP) before the 3rd and 4th doses.

rhGAA and glycogen levels were determined according to the followingsampling protocol. Blood was taken from each study animal after the 4thdose for plasma pharmacokinetic analysis. GAA activity, Western blots,and 1-DNJ levels were determined for plasma samples taken 2 and 4 hoursafter the last SQ dose.

Tissue samples were collected to determine rhGAA uptake 3 days followingthe last dose of the study (i.e., 3 days post dose 4). Tissue samplesincluded heart, diaphragm, tongue, brain, spleen, liver, biceps,triceps, quadriceps, soleus, gastrocnemius, ventral skin and dorsal skinfrom the SQ injection site. Tissue samples were also collected todetermine glycogen concentration 14 days following the last dose of thestudy (i.e., 14 days post dose 4). A summary of the treatmentsadministered to the test subjects, and the timing of sample collectionis shown in Table 2.

TABLE 2 Treatments administered and sample collection times of thepresent study. Lumizyme ® Co-formulation of alone SQ, Lumizyme ® + 1-Plasma Necropsy Group mg/kg DNJ SQ, mg/kg timepoint, hr day 1 0 0 2 3 20 0 4 14 3 20 0 2 3 4 20 0 4 14 5 0 20 L + 30 1-DNJ 2 3 6 0 20 L + 301-DNJ 4 14

Dosing was administered each Monday and Thursday for 2 weeks for a totalof 4 administrations. There were no deaths in any group.

Results

Tissue rhGAA and Glycogen

FIGS. 15-25 show GAA activity 3 days following the final treatment dose,and glycogen levels 14 days following the final treatment dose in tissuesamples taken from animals receiving one of the three treatments. Formost of the tissues tested, co-formulating rhGAA with 1-DNJsignificantly increased rhGAA uptake and activity in the tissues testedcompared to administering rhGAA alone. Additionally, following treatmentwith rhGAA, or the co-formulation of rhGAA and 1-DNJ, rhGAA activity washigh at the site of SQ injection, in ventral skin and in forelimbmuscles 3 days following the final treatment dose. rhGAA activity washigher in liver than in all muscles tested except for forelimbs.

Furthermore, as described in Table 3, rhGAA levels achieved with theco-formulation of 1-DNJ and rhGAA (Lumizyme®) administered SQ wereeither as high or higher (in a majority of tissues) compared to levelsachieved by administering rhGAA (Myozyme®) intravenously alone.

TABLE 3 rhGAA activity uptake (nmol/mg/hr) of a co-formulation of rhGAA(Lumizyme ®) and 1-DNJ administered SQ compared to rhGAA (Myozyme ®)administered alone intravenously (IV) and Lumizyme ® administered aloneSQ. Lumizyme ® + Myozyme ® Lumizyme ® 1-DNJ co-formulation. Tissue IV¹SQ² SQ² Skin 27.1 75 118 Heart 1.8 1.9 3.0 Diaphragm 7.1 4.2 6.1 Tongue12.4 2.7 5.5 Triceps 3.9 88.0 155 Biceps 10.1 56.0 60 Quadriceps 6.9 2.15.7 Gastrocnemius 17.9 2.0 3.1 Soleus 8.0 2.7 2.2 ¹GAA KO mice weregiven 4 doses of Myozyme (20 mg/kg) IV bolus at 2 week intervals. ²GAAKO mice were given 4 doses of Lumizyme (20 mg/kg) +/− 1-DNJ (30 mg/kg)SQ at 3-4 day intervals (Mon and Thu). Enzyme activity uptake data forboth studies assessed 3 days after the last (4th) dose.

With regard to the glycogen levels detected in the tissue samples, themagnitude of reduction in glycogen was correlated with rhGAA uptake insome tissues. Heart, tongue, and ventral skin showed a correlationbetween improved uptake of rhGAA and improved glycogen reduction withthe rhGAA+1-DNJ co-formulation over rhGAA alone. Without being bound byany theory, high rhGAA enzyme activity in some tissue lysates could befrom enzyme that is in the tissue (e.g., fat, lymph, blood vessels) butthat has not yet been taken into the cells and lysosomes. Additionally,without being bound by any theory, there may be an abundance ofcytoplasmic glycogen that is not available to the lysosomal enzyme butis detectable in whole cell lysates.

Plasma rhGAA

To determine whether 1-DNJ present in plasma samples from animalstreated with the co-formulation inhibited rhGAA in the samples, rhGAAactivity was determined by incubating rhGAA from the plasma samples withGAA substrate for 1 hour, and determining enzyme activity based onsubstrate metabolism. Samples were then re-analyzed by incubating thesamples for 3 hours with substrate to allow for dissociation of 1-DNJfrom the enzyme, and thereby reverse any enzyme inhibition caused by1-DNJ. No substantial changes were seen in results between the two assayformats.

FIGS. 26, 27A, 27B, 28A, and 28B show plasma rhGAA activity and proteinconcentration in samples collected 2 and 4 hours following the finaltreatment dose. 2 hours after the final SQ treatment dose, higher rhGAAactivity and protein concentrations were detectable in plasma (i.e.,activity assay and Western blot, respectively) when 1-DNJ wasadministered with rhGAA in the co-formulation than when rhGAA wasadministered alone. 4 hours after the final SQ treatment dose, theplasma rhGAA levels remained elevated in the mice treated with theco-formulation, while plasma rhGAA levels from mice treated with rhGAAalone increased compared to the levels in the 2 hour samples.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions,GenBank Accession Numbers, and protocols are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

1-20. (canceled)
 21. A method of treating Pompe disease in a subjectcomprising administering (i) a first composition comprising an activesite-specific chaperone selected from the group consisting of1-deoxynojirimycin or a pharmaceutically acceptable salt thereof andn-butyl-deoxynojirimycin or a pharmaceutically acceptable salt thereof,and (ii) a second composition comprising acid α-glucosidase, wherein theacid α-glucosidase is formulated at a concentration of about 5 mg/mL toabout 80 mg/mL in the second composition, and wherein the activesite-specific chaperone is administered in an amount of about 1 mg/kg toabout 10 mg/kg and the acid α-glucosidase is administered in an amounteffective for treating Pompe disease in the subject.
 22. The method ofclaim 21, wherein the acid α-glucosidase is formulated at aconcentration of about 15 mg/mL in the second composition.
 23. Themethod of claim 21, wherein the acid α-glucosidase is administered in anamount of about 5 mg/kg to about 50 mg/kg.
 24. The method of claim 23,wherein the acid α-glucosidase is administered in an amount of about 10mg/kg to about 40 mg/kg.
 25. The method of claim 24, wherein the acidα-glucosidase is administered in an amount of about 15 mg/kg to about 25mg/kg.
 26. The method of claim 25, wherein the acid α-glucosidase isadministered in an amount of about 20 mg/kg.
 27. The method of claim 21,wherein at least one of the first composition and the second compositionfurther comprises an excipient.
 28. The method of claim 21, wherein thesecond composition further comprises a buffer.
 29. The method of claim28, wherein the buffer is selected from the group consisting of citratebuffer, acetate buffer, bicarbonate buffer, phosphate buffer, andcombinations thereof.
 30. The method of claim 21, wherein the activesite-specific chaperone is n-butyl-deoxynojirimycin or apharmaceutically acceptable salt thereof.
 31. The method of claim 21,wherein the active site-specific chaperone is 1-deoxynojirimycin or apharmaceutically acceptable salt thereof.
 32. The method of claim 21,wherein the first composition is administered orally and the secondcomposition is administered intravenously.
 33. A method of treatingPompe disease in a subject comprising administering (i) a firstcomposition comprising n-butyl-deoxynojirimycin or a pharmaceuticallyacceptable salt thereof, and (ii) a second composition comprising acidα-glucosidase, wherein the acid α-glucosidase is formulated at aconcentration of about 15 mg/mL in the second composition; and whereinthe n-butyl-deoxynojirimycin or pharmaceutically acceptable salt thereofis administered in an amount of about 1 mg/kg to about 10 mg/kg and theacid α-glucosidase is administered in an amount of about 5 mg/kg toabout 50 mg/kg.
 34. The method of claim 33, wherein the acidα-glucosidase is administered in an amount of about 10 mg/kg to about 40mg/kg.
 35. The method of claim 34, wherein the acid α-glucosidase isadministered in an amount of about 15 mg/kg to about 25 mg/kg.
 36. Themethod of claim 35, wherein the acid α-glucosidase is administered in anamount of about 20 mg/kg.
 37. The method of claim 33, wherein at leastone of the first composition and the second composition furthercomprises an excipient.
 38. The method of claim 33, wherein the secondcomposition further comprises a buffer.
 39. The method of claim 38,wherein the buffer is selected from the group consisting of citratebuffer, acetate buffer, bicarbonate buffer, phosphate buffer, andcombinations thereof.
 40. The method of claim 33, wherein the firstcomposition is administered orally and the second composition isadministered intravenously.